US9617532B2 - Lysis, binding and/or wash reagent for isolating and/or purifying nucleic acids - Google Patents

Lysis, binding and/or wash reagent for isolating and/or purifying nucleic acids Download PDF

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US9617532B2
US9617532B2 US12/994,308 US99430809A US9617532B2 US 9617532 B2 US9617532 B2 US 9617532B2 US 99430809 A US99430809 A US 99430809A US 9617532 B2 US9617532 B2 US 9617532B2
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polyoxyethylene
lysis
binding
ether
reagent
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US20110076751A1 (en
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Roland Fabis
Anke Homann-Wischinski
Thorsten Voss
Thomas Hanselle
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Qiagen GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to a lysis, binding and/or wash reagent and to a method for isolating and/or purifying nucleic acids.
  • Said lysis, binding and/or wash reagent and method are particularly suitable for use purposes in molecular diagnostics.
  • the prior art has disclosed a multiplicity of methods for isolating and/or purifying nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) from cells, cell cultures or virus cultures.
  • nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) from cells, cell cultures or virus cultures.
  • “classical” methods for isolating nucleic acids are based on a one-step method which involves carrying out an extraction after the addition of an aqueous buffer and an organic extractant.
  • the nucleic acids remain in the aqueous phase and may be isolated after the organic phase which contains undesired accompanying substances has been removed.
  • nucleic acids to solid, mostly mineral supports such as silicon dioxide.
  • the binding principle is based on a reversible binding of the nucleic acids under the influence of “chaotropic” salts and/or alcohol to the silicon dioxide surface.
  • various solutions or mixtures usually lysis, binding, wash and/or eluting solutions or mixtures, are added to the nucleic acid-containing sample, and in a final method step the purified nucleic acid is eluted from the support which has been added in the binding step at the latest.
  • the basic principle of both methods is based on the cells, in particular the plant, animal, human, bacterial or viral cells, being lysed in a first step. To this end, the cells are firstly incubated with a lysis buffer which disrupts the cells.
  • lysis buffers frequently contain the surfactant polyoxyethylene sorbitan monolaurate (Tween® 20). This surfactant is used for converting contaminations into a soluble or stabilized state during cell lysis, in order to remove them from the nucleic acid.
  • Teween® 20 polyoxyethylene sorbitan monolaurate
  • lysis buffers containing polyoxyethylene sorbitan monolaurate are not stable during storage. For example, the pH decreases.
  • a particular disadvantage is the fact that the yield of the isolated nucleic acids is reduced after storage when these lysis buffers are used.
  • Another disadvantage is the fact that the eluates containing nucleic acids are opaque, indicating the presence of contaminations which may interfere with further usage of the isolated nucleic acids.
  • a lysis, binding and/or wash reagent comprising:
  • lysis, binding and/or wash reagent means for the purposes of the present invention reagents that are lysis reagents, binding reagents or wash reagents, and also reagents which may act as lysis reagent and also as binding reagent and also as wash reagent. More specifically, the term “lysis, binding and/or wash reagent” means for the purposes of the present invention also mixtures of lysis reagents, binding reagents and/or wash reagents of the invention.
  • reagent means for the purposes of the present invention lysis, binding and/or wash reagent.
  • chaotropic compound means for the purposes of the present invention compounds which act in a denaturing fashion on proteins and which in particular destroy the regular structure of liquid water, which is based on the formation of hydrogen bonds.
  • buffer compound means for the purposes of the present invention compounds which may provide buffering or pH stabilization of an aqueous solution.
  • phosphate buffer means for the purposes of the present invention phosphate salts such as dihydrogen phosphates, for example potassium dihydrogen phosphate (KH 2 PO 4 ) or sodium dihydrogen phosphate (NaH 2 PO 4 ), and hydrogen phosphates, for example disodium hydrogen phosphate dihydrate (Na 2 HPO 4 .2H 2 O) or dipotassium hydrogen phosphate. Mixtures of the phosphate salts may likewise be used.
  • PBS phosphate buffered saline
  • PBS phosphate buffered saline
  • nucleic acid means for the purposes of the present invention—but is not limited to—natural, preferably isolated, linear, branched or circular nucleic acids such as RNA, in particular mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA or ribozymes, DNA, plasmid DNA and the like, synthetic or modified nucleic acids, in vitro transcripts, for example oligonucleotides, more specifically primers, probes or standards usable for PCR, nucleic acids labelled with digoxygenin, biotin or fluorescent dyes, methylated nucleic acids or “PNAs” (“peptide nucleic acids”).
  • RNA in particular mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA or ribozymes, DNA, plasmid DNA and the like
  • synthetic or modified nucleic acids in vitro transcripts, for example oligonucleotides, more specifically primers, probes or standards usable for PCR, nucle
  • surfactant means for the purposes of the present invention interface-active and/or surface-active substances.
  • fatty alcohol means for the purposes of the present invention alcohols having a chain length of from 6 to 22 carbon atoms, preferably 8 to 20 carbon atoms, preferentially 10 to 18 carbon atoms, particularly preferably 12 to 18 carbon atoms. Preference is in particular given to alcohols having 12, 14, 16 or 18 carbon atoms. Although the fatty alcohols may be mono- or polyunsaturated, they are preferably saturated fatty alcohols.
  • Polyoxyethylene means for the purposes of the present invention an HO—(CH 2 CH 2 O) n unit, with n being preferably an integer from 2 to 150, further preferably from 4 to 120, still further preferably from 8 to 80, and most preferably an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
  • Polyoxypropylene means for the purposes of the present invention an HO—(CH 2 CH 2 CH 2 O) n unit, with n being preferably an integer from 10 to 90, further preferably from 20 to 80, still further preferably from 30 to 70, and most preferably n being an integer selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.
  • % weight/volume means for the purposes of the present invention the information of grams of surfactant per 100 ml of reagent or composition, for example.
  • lysis, binding and/or wash reagents of the invention were found to have improved stability during storage.
  • lysis, binding and/or wash reagents of the invention may have a stable pH during storage at room temperature for three, preferably six, months, further preferably at least eight months.
  • lysis, binding and/or wash reagents of the invention may also have a stable pH when stored at elevated temperatures, for example at 50° C., for several weeks, preferably for several months.
  • Polyoxyethylene-based non-ionic surfactants preferred according to the invention are polyoxyethylene fatty alcohol ethers.
  • polyoxyethylene fatty alcohol ethers examples include polyethoxylated lauryl, cetyl, oleyl, or stearyl alcohols which may be used alone or as mixture.
  • the polyoxyethylene fatty alcohol ether comprises a fatty alcohol component having from 6 to 22 carbon atoms and a polyoxyethylene component having from 2 to 150 (CH 2 CH 2 O) units.
  • the polyoxyethylene fatty alcohol ether is selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and/or polyoxyethylene oleyl ether.
  • Polyoxyethylene-based non-ionic surfactants in particular polyoxyethylene fatty alcohol ethers, have turned out to be advantageous for a broad range of applications within the present invention. Improved stability during storage can be observed in particular with lysis, binding and/or wash reagents comprising buffer compounds preferably selected from the group comprising tris(hydroxymethyl)aminomethane (TRIS), N-(tri(hydroxymethyl)methyl)glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (BICINE), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (HEPES), piperazine-1,4-bis(2-ethanesulphonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulphonic acid (CHES), 2-(N-morpholino)ethanesulphonic acid (MES), 3-(N-morpholino)propanesulphonic acid (MOPS) and/or phosphate buffer.
  • the lysis, binding and/or wash reagents of the invention were found to have advantageous effects in particular with a content of polyoxyethylene-based non-ionic surfactant selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers in the range from ⁇ 8% (weight/volume) to ⁇ 50% (weight/volume), based on the total volume of the lysis, binding and/or wash reagent.
  • a content of polyoxyethylene-based non-ionic surfactant selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers in the range from ⁇ 8% (weight/volume) to ⁇ 50% (weight/volume), based on the total volume of the lysis, binding and/or wash reagent.
  • the concentration information is preferably the total surfactant content, for example in the range from ⁇ 8% (weight/volume) to ⁇ 50% (weight/volume), based on the total volume of the reagent.
  • Preferred polyoxyethylene fatty alcohol ethers are ethoxylated lauryl, cetyl, oleyl or stearyl alcohols selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and/or polyoxyethylene oleyl ether.
  • Preferred polyoxyethylene fatty alcohol ethers are selected from the group comprising polyoxyethylene(4) lauryl ether, polyoxyethylene(23) lauryl ether, polyoxyethylene(2) cetyl ether, polyoxyethylene(10) cetyl ether, polyoxyethylene(20) cetyl ether, polyoxyethylene(2) stearyl ether, polyoxyethylene(10) stearyl ether, polyoxyethylene(20) stearyl ether, polyoxyethylene(2) oleyl ether, polyoxyethylene(10) oleyl ether, polyoxyethylene(20) oleyl ether and/or polyoxyethylene(100) stearyl ether.
  • the numbers here indicate the average number of ethylene oxide units.
  • Particularly suitable according to the invention are polyoxyethylene fatty alcohol ethers sold under the trade name Brij®, for example by ICI Surfactants.
  • polyoxyethylene lauryl, polyoxyethylene cetyl, polyoxyethylene oleyl or polyoxyethylene stearyl alcohol ethers are preferably selected from the group comprising polyoxyethylene(4) lauryl ether (Brij® 30), polyoxyethylene(23) lauryl ether (Brij® 35), polyoxyethylene(2) cetyl ether (Brij® 52), polyoxyethylene(10) cetyl ether (Brij® 56), polyoxyethylene(20) cetyl ether (Brij® 58), polyoxyethylene(2) stearyl ether (Brij® 72), polyoxyethylene(10) stearyl ether (Brij® 76), polyoxyethylene(20) stearyl ether (Brij® 78), polyoxyethylene(2) oleyl ether (Brij® 92), polyoxyethylene(10) oleyl ether (Brij® 97), polyoxyethylene(20) oleyl ether (Brij® 98) and/or polyoxyethylene(100) stearyl ether (Brij® 700).
  • Suitable polyoxyethylene lauryl, polyoxyethylene cetyl, polyoxyethylene oleyl or polyoxyethylene stearyl alcohol ethers may also be used as powders, for example polyoxyethylene(21) stearyl ether powder (Brij® 721P).
  • lysis, binding and/or wash reagent of the invention may be provided by lysis, binding and/or wash reagents, when used for isolating and/or purifying nucleic acids, showing a consistently good yield of the isolated nucleic acids, even if the lysis, binding and/or wash reagent has been stored at room temperature or at elevated temperatures, for example up to 50° C., for several weeks or several months, while prior art buffers, in particular buffers containing Tween® 20, show lower yields of nucleic acids after storage.
  • a particular advantage of the use of lysis, binding and/or wash reagents of the invention is the fact that an eluate containing the nucleic acids is not or only slightly opaque, even after several weeks or several months of storage. It is therefore an advantage that the eluate may contain no or at least distinctly fewer contaminations, thereby rendering the further use of the eluate containing the nucleic acids substantially more advantageous because further time-consuming purification steps that reduce the yield of nucleic acids may be dispensed with.
  • the lysis, binding and/or wash reagents comprising lauryl alcohol ethers of polyoxyethylene, for example polyoxyethylene(4) lauryl ether (Brij® 30) or polyoxyethylene(23) lauryl ether (Brij® 35). Therefore, in a preferred embodiment, the lysis, binding and/or wash reagents do not contain any of these substances. In a particularly preferred embodiment, the polyoxyethylene fatty alcohol ether of the lysis, binding and/or wash reagent is not polyoxyethylene lauryl ether.
  • the polyoxyethylene fatty alcohol ether is selected from the group comprising polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and/or polyoxyethylene oleyl ether.
  • polyoxyethylene cetyl, polyoxyethylene oleyl, or polyoxyethylene stearyl alcohol ethers selected from the group comprising polyoxyethylene(10) cetyl ether (Brij® 56), polyoxyethylene(20) cetyl ether (Brij® 58), polyoxyethylene(20) stearyl ether (Brij® 78) and/or polyoxyethylene(20) oleyl ether (Brij® 98).
  • polyoxyethylene cetyl or polyoxyethylene oleyl alcohol ethers preferably selected from the group comprising polyoxyethylene(10) cetyl ether (Brij® 56), polyoxyethylene(20) cetyl ether (Brij® 58) and/or polyoxyethylene(20) oleyl ether (Brij® 98).
  • lysis, binding and/or wash reagents comprising polyoxyethylene fatty alcohol ethers, in particular polyoxyethylene cetyl or polyoxyethylene oleyl alcohol ethers, by comparison stand out by a particularly good yield of isolated nucleic acids, in particular viral DNA. More specifically, both use of a freshly prepared lysis and/or binding reagent containing polyoxyethylene cetyl alcohol ether and use after several weeks, in particular several months, of storage at 50° C. for isolating DNA of Hepatitis B virus (HBV) were surprisingly found to produce a markedly increase yield of viral DNA in comparison with lysis buffers containing Tween® 20.
  • HBV Hepatitis B virus
  • lysis and/or binding reagent of the invention may provide in particular a particular advantage of the lysis and/or binding reagent of the invention because Hepatitis B virus (HBV) is known to be difficult to lyse.
  • the lysis reagent of the invention is particularly suitable for isolating viral DNA.
  • lauryl, cetyl, stearyl or oleyl alcohols which are available, for example, under the INCI names laureth, ceteth, steareth or oleth.
  • Examples of furthermore suitable ethoxylated dodecyl, lauryl, cetyl, stearyl or oleyl alcohols are available under names selected from the group comprising laureth-9, laureth-4, laureth-23, ceteth-2, ceteth-20, steareth-2, steareth-10, steareth-20, oleth-2, oleth-10 and/or oleth-20.
  • polyoxyethylene-based non-ionic surfactants are polyoxyethylene alkylphenyl ethers. Preference is given to polyoxyethylene alkylphenyl ethers having an alkyl group containing from five to 15 carbon atoms, preferably containing 6 to 10 carbon atoms. Further preference is given to branched or unbranched C 7 - to C 10 -alkyl groups, more particularly branched or unbranched C 8 - and C 9 -alkyl groups, particularly preferably isooctyl groups and nonyl groups.
  • the polyoxyethylene alkylphenyl ether is selected from the group comprising polyoxyethylene nonylphenyl ether and/or polyoxyethylene isooctylphenyl ether.
  • Suitable polyoxyethylene nonylphenyl ethers and polyoxyethylene isooctylphenyl ethers are obtainable, for example, under the trade name Igepal®, for example from BASF.
  • polyoxyethylene nonylphenyl ethers and polyoxyethylene isooctylphenyl ethers are preferably selected from the group comprising polyoxyethylene(2) nonylphenyl ether (Igepal® CO-210), polyoxyethylene(2) isooctylphenyl ether (Igepal® CA-210), polyoxyethylene(5) nonylphenyl ether (Igepal® CO-520), polyoxyethylene(5) isooctylphenyl ether (Igepal® CA-520), polyoxyethylene(9) nonylphenyl ether (Igepal® CO-630), polyoxyethylene(9) isooctylphenyl ether (Igepal® CA-630), polyoxyethylene(12) nonylphenyl ether (Igepal® CO-720), polyoxyethylene(12) isooctylphenyl ether (Igepal® CA-720) and/or polyoxyethylene(100) nonylphenyl ether (Igepal® CO-210
  • polyoxyethylene-based non-ionic surfactants are polyoxyethylene-polyoxypropylene block copolymers.
  • Polyoxyethylene-polyoxypropylene block copolymers are also referred to as “poloxamers”. Preference is given to polyoxyethylene-polyoxypropylene block copolymers of the empirical formula HO(C 2 H 4 O) a (C 3 H 6 O) b (C 2 H 4 O) a H, where “a” refers to the number of polyoxyethylene units and “b” refers to the number of polyoxypropylene units, with the a/b weight ratio preferably being in the range from 0.1 to 3.
  • “a” is more particularly in the range from 2 to 150, preferably in the range from 4 to 120, further preferably in the range from 8 to 80, still further preferably “a” is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103
  • “b” is more particularly in the range from 10 to 90, preferably in the range from 20 to 80, further preferably in the range from 30 to 70, still further preferably “b” is an integer selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90, and most preferably “b” is an integer selected from 15, 18, 23, 40, 55, 67 and 75.
  • polyoxyethylene-polyoxypropylene block copolymers having polyoxyethylene and polyoxypropylene blocks of different lengths, in which a polyoxypropylene block having from 15 to 67 polypropylene units is enclosed by two polyoxyethylene blocks having independently of one another in each case from 2 to 130 polyethylene units.
  • Suitable polyoxyethylene-polyoxypropylene block copolymers can be obtained, for example, under the trade name Pluronic® or Synperonic®, for example from BASF.
  • suitable polyoxyethylene-polyoxypropylene block copolymers are preferably selected from the group comprising Pluronic® PE 6200, Pluronic® PE 6400, Pluronic® PE 6800, Pluronic® PE 10300, Pluronic® PE 10500, Pluronic® F127, Pluronic® F108, Synperonic® F108, Synperonic® F127 and/or Synperonic® F68.
  • the lysis, binding and/or wash reagent comprises a non-ionic surfactant in the range from ⁇ 9% (weight/volume) to ⁇ 40% (weight/volume), preferably in the range from ⁇ 10% (weight/volume) to ⁇ 30% (weight/volume), preferentially in the range from ⁇ 15% (weight/volume) to ⁇ 20% (weight/volume), based on the total volume of the reagent.
  • the chaotropic compound is a sodium salt or guanidinium salt, preferably selected from the group comprising sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate, and/or a mixture of two or more salts thereof.
  • the chaotropic compound is preferably a guanidinium salt, preferentially selected from the group comprising guanidinium hydrochloride, guanidinium thiocyanate, and/or guanidinium isothiocyanate.
  • a combination of the abovementioned chaotropic compounds and the polyoxyethylene-based non-ionic surfactants has turned out to be advantageous for lysing virus cells and isolating nucleic acids from virus cells.
  • Suitable concentrations and amounts of chaotropic compounds may vary depending on the type of samples or parameters of lysis, with concentrations of the chaotropic compound in the range from ⁇ 0.1 M to ⁇ 10 M, based on the total volume of the reagent, being generally advantageous. Preference is given to the concentrations of the chaotropic compound of the lysis, binding and/or wash reagent being in the range from ⁇ 0.5 M to ⁇ 8 M, preferably in the range from ⁇ 0.9 M to ⁇ 6 M.
  • the concentrations of the chaotropic compound of the lysis reagent are preferably in the range from ⁇ 3 M to ⁇ 7 M, particularly preferably in the range from ⁇ 4 M to ⁇ 6 M.
  • the concentrations of the chaotropic compound of the binding reagent are preferably in the range from ⁇ 0.5 M to ⁇ 7 M, particularly preferably in the range from ⁇ 1 M to ⁇ 6 M.
  • the concentrations of the chaotropic compound of the wash reagent are preferably in the range from ⁇ 0.5 M to ⁇ 3.5 M, particularly preferably in the range from ⁇ 0.9 M to ⁇ 3 M.
  • the lysis, binding and/or wash reagent comprises at least one buffer compound selected from the group comprising tris(hydroxymethyl)aminomethane (TRIS), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (HEPES), 3-(N-morpholino)propanesulphonic acid (MOPS) and/or phosphate buffer.
  • TMS tris(hydroxymethyl)aminomethane
  • HPES N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid)
  • MOPS 3-(N-morpholino)propanesulphonic acid
  • the lysis, binding and/or wash reagent comprises at least one buffer compound selected from the group comprising tris(hydroxymethyl)aminomethane (TRIS) and/or N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (HEPES).
  • TMS tris(hydroxymethyl)aminomethane
  • HEPES N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid)
  • the lysis, binding and/or wash reagent is preferably an aqueous solution.
  • the lysis, binding and/or wash reagent has a pH in the range from ⁇ 4 to ⁇ 12, more particularly in the range from ⁇ 6 to ⁇ 11, preferably in the range from ⁇ 7 to ⁇ 10, particularly preferably in the range from ⁇ 8 to ⁇ 9.
  • the lysis, binding and/or wash reagent in particular the lysis reagent, may furthermore have enzymes, for example lytic enzymes, in particular, for example, proteinase K, protease (e.g. QIAGEN Protease), zymolase, lyticase, achromopeptidase, lysostaphin, lysozyme and, depending on use, nucleases, for example DNase and/or RNase.
  • enzymes for example lytic enzymes, in particular, for example, proteinase K, protease (e.g. QIAGEN Protease), zymolase, lyticase, achromopeptidase, lysostaphin, lysozyme and, depending on use, nucleases, for example DNase and/or RNase.
  • Lysis, binding and/or wash reagents of the invention may be lysis reagents, binding reagents or wash reagents, or mixtures of lysis reagents, binding reagents and/or wash reagents of the invention.
  • Nucleic acids are immobilized on a matrix based on one or more silicon oxide compounds in the presence of a chaotropic compound preferentially in the presence of a branched or unbranched alkanol. Preference is therefore given to the binding reagent comprising at least one branched or unbranched alkanol.
  • the branched or unbranched alkanol is an alcohol having from one to five carbon atoms, preferably selected from the group comprising methanol, ethanol, isopropanol, n-propanol, branched or unbranched butanol or pentanol, and/or mixtures thereof.
  • branched or unbranched alkanol in particular propanol, butanol and pentanol comprise any consumable isomeric forms of the particular radicals.
  • branched or unbranched propanol comprises n-propanol and isopropanol
  • branched or unbranched butanol comprises isobutanol, sec-butanol and tert-butanol
  • branched or unbranched pentanol comprises, for example, n-pentanol and isopentanol.
  • the binding reagent comprises branched or unbranched alkanol in the range from ⁇ 20% by volume to ⁇ 80% by volume, preferentially in the range from ⁇ 40% by volume to ⁇ 70% by volume, preferably in the range from ⁇ 50% by volume to ⁇ 60% by volume, based on the total volume of the binding reagent.
  • volume and/or weight contents are specified, it is self-evident to a person skilled in the art that said volume and/or weight contents of the individual components are chosen in such a way that the total volume or total weight of the components does not exceed 100% by volume or 100% by weight.
  • the present invention moreover relates to the use of a lysis, binding and/or wash reagent of the invention for isolating and/or purifying nucleic acids.
  • the present invention furthermore relates to a method for isolating and/or purifying nucleic acids from a nucleic acid-containing biological sample, comprising the following method steps:
  • composition means for the purposes of the present invention lysis and/or binding composition.
  • a lysis reagent of the invention is used for lysing the sample.
  • the lysis reagent is brought into contact with the biological sample to be lysed.
  • One or more enzymes may be added independently of one another at various time points, depending on the use.
  • the sample may be in liquid form, for example in the case of liquid clinical samples.
  • Clinical samples which contain solid components, such as stool samples or swab samples are usually suspended in suitable aqueous solutions prior to further analysis. Cell cultures are usually removed from the culture medium before lysis, but drying the sample completely is avoided in most cases. In the case of completely dried samples, for example lyophilizates, the sample is reconstituted in aqueous solutions prior to further processing, for example lyophilizates of virus standards.
  • the samples to be lysed therefore usually contain some liquid. This liquid present in the sample is brought into contact with the lysis reagent.
  • a method for isolating and/or purifying nucleic acids from a sample usually involves a lysis composition which contains lysis reagent and other liquid of the sample or of solutions already added to said sample.
  • lysis and/or binding composition refers for the purposes of the present invention to a lysis and/or binding reagent which is used in a method for isolating and/or purifying nucleic acids from a sample and which may contain further liquid in addition to lysis, binding and/or wash reagent.
  • the lysis and/or binding composition may preferably comprise lysis and/or binding reagent of the invention.
  • the released nucleic acid is immobilized on a matrix based on one or more silicon oxide compound in the presence of a binding composition of the invention.
  • the lysis composition or another solution in which lysis was carried out may be removed before the sample is brought into contact with the binding reagent. Preference is given to not removing the lysis composition.
  • a binding reagent is brought into contact with a sample comprising lysis composition.
  • lysing is carried out in the presence of a lysis composition and immobilizing is carried out in the presence of a binding composition.
  • immobilizing is preferably carried out in the presence of a mixture of a lysis composition and binding composition.
  • the lysis reagent may also be employed as binding reagent at the same time. Further optionally, the binding reagent may also be employed as lysis reagent. Also optionally, the binding reagent may also be employed as wash reagent.
  • the lysis and/or binding composition comprises at least one chaotropic compound and at least one polyoxyethylene-based non-ionic surfactant selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether and/or polyoxyethylene-polyoxypropylene block copolymers in the range from ⁇ 0.1% (weight/volume) to ⁇ 50% (weight/volume), based on the total volume of the composition. If mixtures of surfactants are used, preferably the total surfactant content is, for example, in the range from ⁇ 0.1% (weight/volume) to ⁇ 50% (weight/volume) in total, based on the total volume of the composition.
  • the advantage of such a method for isolating and/or purifying nucleic acids from a nucleic acid-containing biological sample is that of an eluate containing the nucleic acids being not or only slightly opaque, even after several weeks or several months of storage at room temperature or elevated temperatures, for example, 50° C., when a lysis and/or binding composition comprising at least one chaotropic compound and at least one polyoxyethylene-based non-ionic surfactant selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether and/or polyoxyethylene-polyoxypropylene block copolymers in the range from ⁇ 0.1% (weight/volume) to ⁇ 50% (weight/volume), based on the total volume of the composition, is used.
  • the eluate may contain no or at least distinctly fewer contaminations. This renders further use of the eluate containing the nucleic acids substantially more advantageous, because further time-consuming purification steps that reduce the yield of nucleic acids may be dispensed with.
  • Another advantage of such a method for isolating and/or purifying nucleic acids from a nucleic acid-containing biological sample is that of making possible a particularly good yield of isolated nucleic acids, in particular viral DNA, for example the DNA of Hepatitis B virus (HBV).
  • isolated nucleic acids in particular viral DNA, for example the DNA of Hepatitis B virus (HBV).
  • HBV Hepatitis B virus
  • a “biological sample” may be understood as meaning a material on a particulate or molecular basis, in particular viruses, phages and cells such as bacterial cells, yeast or mould cells or human, animal or plant cells.
  • the method is particularly suitable for isolating nucleic acids such as DNA or RNA from sample materials of human or animal origin, for example clinical samples such as blood, plasma, serum, mouth, throat and nose rinses, bronchoalveolar lavages, urine, cerebral fluid, sputum, saliva, stool, aspirates, smears/swabs such as, for example, nasal smears/swabs, buccal smears/swabs, cervical smears/swabs, vaginal smears/swabs, urethral smears/swabs, pharyngeal smears/swabs, perineal smears/swabs, and rectal smears/swabs, stool, as
  • the sample can also be from the field of environmental analysis, food analysis, or molecular biological research, for example from bacterial cultures, yeast or fungal cultures, viral cultures, phage lysates or products of amplification processes, for example of a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the method of the invention is preferably suitable for isolating and/or purifying genomic DNA, mitochondrial DNA, plasmid DNA, viral DNA and viral RNA, and for isolating and purifying intracellular RNA from whole blood, for example for reverse transcription polymerase chain reaction (RT-PCR), and also for isolating and/or purifying freely circulating nucleic acids present in cell-free sample materials.
  • the method of the invention is particularly suitable for isolating and/or purifying viral DNA.
  • the biological sample is lysed in step a) of the method.
  • the methods listed below selected from the group comprising lysis with the aid of ionic and non-ionogenic surfactants, for example sodium dodecyl sulphate (SDS), lithium dodecyl sulphate (LiDS) or sodium lauroyl sarcosinate (sarkosyl) in suitable reagents or buffers, the use of chaotropic salts, mechanical tearing apart, for example by means of ultrasound, a “French press”, grinding with particles such as glass balls, ceramic balls or metal particles, or in liquid nitrogen, by repeated cycles of freezing and thawing, or by boiling, enzymatic lysis, lysis by freeze-drying, lysis by osmotic shock, microwave and/or temperature treatment, and/or combinations thereof, are suitable for lysing a biological sample. Lysis is preferably carried out in the presence of chaotropic salts.
  • Preference is given to lysing the biological sample in the presence of a lysis composition comprising at least one chaotropic compound and at least one polyoxyethylene-based non-ionic surfactant selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether and/or polyoxyethylene-polyoxypropylene block copolymers in the range from ⁇ 0.1% (weight/volume) to ⁇ 50% (weight/volume), based on the total volume of the lysis composition.
  • a combination of chaotropic agents and polyoxyethylene-based non-ionic surfactant selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers is particularly effective in the lysis of virus cells.
  • the lysis and/or binding composition comprises at least one chaotropic compound and at least one polyoxyethylene-based non-ionic surfactant selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers.
  • ethoxylated fatty alcohols examples include ethoxylated dodecyl, lauryl, cetyl, oleyl or stearyl alcohols which may be used alone or as a mixture.
  • Preferred polyoxyethylene fatty alcohol ethers are ethoxylated lauryl, cetyl, oleyl or stearyl alcohols selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and/or polyoxyethylene oleyl ether.
  • Preferred polyoxyethylene fatty alcohol ethers are selected from the group comprising polyoxyethylene(4) lauryl ether, polyoxyethylene(23) lauryl ether, polyoxyethylene(2) cetyl ether, polyoxyethylene(10) cetyl ether, polyoxyethylene(20) cetyl ether, polyoxyethylene(2) stearyl ether, polyoxyethylene(10) stearyl ether, polyoxyethylene(20) stearyl ether, polyoxyethylene(2) oleyl ether, polyoxyethylene(10) oleyl ether, polyoxyethylene(20) oleyl ether, and/or polyoxyethylene(100) stearyl ether.
  • the numbers here indicate the average number of ethylene oxide units.
  • polyoxyethylene lauryl, polyoxyethylene cetyl, polyoxyethylene oleyl, or polyoxyethylene stearyl alcohol ethers are preferably selected from the group comprising polyoxyethylene(4) lauryl ether (Brij® 30) or polyoxyethylene(23) lauryl ether (Brij® 35), polyoxyethylene(2) cetyl ether (Brij® 52), polyoxyethylene(10) cetyl ether (Brij® 56), polyoxyethylene(20) cetyl ether (Brij® 58), polyoxyethylene(2) stearyl ether (Brij® 72), polyoxyethylene(10) stearyl ether (Brij® 76), polyoxyethylene(20) stearyl ether (Brij® 78), polyoxyethylene(2) oleyl ether (Brij® 92), polyoxyethylene(10) oleyl ether (Brij® 97), polyoxyethylene(20) oleyl ether (Brij® 98), and/or polyoxyethylene(100) stearyl ether (Brij® 700
  • the polyoxyethylene fatty alcohol ether comprises a fatty alcohol component having from 6 to 22 carbon atoms and a polyoxyethylene component containing from 2 to 150 (CH 2 CH 2 O) units.
  • the polyoxyethylene fatty alcohol ether is selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and/or polyoxyethylene oleyl ether.
  • the polyoxyethylene fatty alcohol ether is selected from the group comprising polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and/or polyoxyethylene oleyl ether.
  • the lysis and/or binding composition comprising lauryl alcohol ethers of polyoxyethylene, for example polyoxyethylene(4) lauryl ether (Brij® 30) or polyoxyethylene(23) lauryl ether (Brij® 35). Preference is therefore given to the lysis and/or binding composition not containing any of these substances.
  • the polyoxyethylene fatty alcohol ether of the lysis and/or binding composition is not polyoxyethylene lauryl ether.
  • polyoxyethylene cetyl, polyoxyethylene oleyl, or polyoxyethylene stearyl alcohol ether preferably selected from the group comprising polyoxyethylene(10) cetyl ether (Brij® 56), polyoxyethylene(20) cetyl ether (Brij® 58), polyoxyethylene(20) stearyl ether (Brij® 78) and/or polyoxyethylene(20) oleyl ether (Brij® 98).
  • polyoxyethylene cetyl or polyoxyethylene oleyl alcohol ethers preferably selected from the group comprising polyoxyethylene(10) cetyl ether (Brij® 56), polyoxyethylene(20) cetyl ether (Brij® 58) and/or polyoxyethylene(20) oleyl ether (Brij® 98).
  • lauryl, cetyl, stearyl or oleyl alcohols which are available, for example, under the INCI names laureth, ceteth, steareth or oleth.
  • polyoxyethylene-based non-ionic surfactants are polyoxyethylene alkylphenyl ethers. Preference is given to polyoxyethylene alkylphenyl ethers having an alkyl group containing from five to 15 carbon atoms, preferably containing 6 to 10 carbon atoms. Further preference is given to branched or unbranched C 7 - to C 10 -alkyl groups, more particularly branched or unbranched C 8 - and C 9 -alkyl groups, particularly preferably isooctyl groups and nonyl groups.
  • the polyoxyethylene alkylphenyl ether is selected from the group comprising polyoxyethylene nonylphenyl ether and/or polyoxyethylene isooctylphenyl ether.
  • Suitable polyoxyethylene nonylphenyl ethers and polyoxyethylene isooctylphenyl ethers are obtainable, for example, under the trade name Igepal®, for example from BASF.
  • polyoxyethylene-based non-ionic surfactants are polyoxyethylene-polyoxypropylene block copolymers.
  • Polyoxyethylene-polyoxypropylene block copolymers are also referred to as “poloxamers”. Preference is given to polyoxyethylene-polyoxypropylene block copolymers of the empirical formula HO(C 2 H 4 O) a (C 3 H 6 O) b (C 2 H 4 O) a H, where “a” refers to the number of polyoxyethylene units and “b” refers to the number of polyoxypropylene units, with the a/b weight ratio preferably being in the range from 0.1 to 3.
  • “a” is more particularly in the range from 2 to 150, preferably from 4 to 120, further preferably from 8 to 80, still further preferably “a” is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105
  • “b” is more particularly in the range from 10 to 90, preferably in the range from 20 to 80, further preferably in the range from 30 to 70, still further preferably “b” is an integer selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90, and most preferably “b” is an integer selected from 15, 18, 23, 40, 55, 67 and 75.
  • polyoxyethylene-polyoxypropylene block copolymers having polyoxyethylene and polyoxypropylene blocks of different lengths, in which a polyoxypropylene block having from 15 to 67 polypropylene units is enclosed by two polyoxyethylene blocks having independently of one another in each case from 2 to 130 polyethylene units.
  • Suitable polyoxyethylene-polyoxypropylene block copolymers can be obtained, for example, under the trade name Pluronic® or Synperonic®, for example from BASF.
  • the lysis and/or binding composition comprises a non-ionic surfactant in the range from ⁇ 0.2% (weight/volume) to ⁇ 30% (weight/volume), preferably in the range from ⁇ 3% (weight/volume) to ⁇ 10% (weight/volume), preferentially in the range from ⁇ 3.2% (weight/volume) to ⁇ 8% (weight/volume), based on the total volume of the composition.
  • the chaotropic compound of the lysis and/or binding composition is a sodium salt or guanidinium salt, preferably selected from the group comprising sodium iodide, sodium perchlorate, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate, and/or a mixture of two or more salts thereof.
  • the chaotropic compound is preferably a guanidinium salt, preferentially selected from the group comprising guanidinium hydrochloride, guanidinium thiocyanate, and/or guanidinium isothiocyanate.
  • a combination of the abovementioned chaotropic compounds and the polyoxyethylene-based non-ionic surfactants has turned out to be advantageous for lysing virus cells and isolating nucleic acids from virus cells.
  • Concentrations of the chaotropic compound of the lysis and/or binding composition in the range from ⁇ 0.1 M to ⁇ 10 M have turned out to be advantageous. Preference is given to the concentrations of the chaotropic compound in the range from ⁇ 1 M to ⁇ 8 M, preferably in the range from ⁇ 3 M to ⁇ 7 M, particularly preferably in the range from ⁇ 4 M to ⁇ 6 M.
  • the lysis and/or binding composition comprises at least one buffer compound selected from the group comprising tris(hydroxymethyl)aminomethane (TRIS), N-(tri(hydroxymethyl)methyl)glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine,(BICINE), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (HEPES), piperazine-1,4-bis(2-ethanesulphonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulphonic acid (CHES), 2-(N-morpholino)ethanesulphonic acid (MES), 3-(N-morpholino)propanesulphonic acid (MOPS) and/or phosphate buffer.
  • TMS tris(hydroxymethyl)aminomethane
  • Tricine N-(tri(hydroxymethyl)methyl)glycine
  • BICINE N,N-bis(2-hydroxyethyl)glycine
  • the lysis and/or binding composition comprises at least one buffer compound selected from the group comprising tris(hydroxymethyl)aminomethane (TRIS), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (HEPES) and/or phosphate buffer.
  • the lysis and/or binding composition comprises at least one buffer compound selected from the group comprising tris(hydroxymethyl)aminomethane (TRIS) and/or N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (HEPES).
  • the biological sample may be lysed at room temperature, for example at 15° C. to 25° C., or at an elevated temperature, for example at temperatures in the range from ⁇ 37° C. to ⁇ 75° C.
  • the lysis composition may furthermore have enzymes, for example proteinase K, protease (e.g. QIAGEN Protease), zymolase, lyticase, achromopeptidase, lysostaphin, lysozyme and, depending on use, nucleases, for example DNase and/or RNase.
  • enzymes for example proteinase K, protease (e.g. QIAGEN Protease), zymolase, lyticase, achromopeptidase, lysostaphin, lysozyme and, depending on use, nucleases, for example DNase and/or RNase.
  • the released nucleic acid(s) is(are) immobilized on a matrix based on one or more silicon oxide compound in the presence of a chaotropic compound and/or a branched or unbranched alkanol.
  • the binding composition comprising a branched or unbranched alkanol.
  • the branched or unbranched alkanol is an alcohol having from one to five carbon atoms, preferably selected from the group comprising methanol, ethanol, isopropanol, n-propanol, n-butanol, isobutanol, sec-butanol, tert-butanol, n-pentanol, isopentanol, and/or mixtures thereof.
  • the binding composition comprises branched or unbranched alkanol in the range from ⁇ 1% by volume to ⁇ 80% by volume, preferentially in the range from ⁇ 5% by volume to ⁇ 70% by volume, preferably in the range from ⁇ 10% by volume to ⁇ 60% by volume, further preferably in the range from ⁇ 15% by volume to ⁇ 50% by volume, based on the total volume of the binding composition.
  • a mixture of the binding composition comprises the lysis reagent and optionally one or more further additives, preferably branched or unbranched alkanol in the range from ⁇ 1% by volume to ⁇ 80% by volume, preferentially in the range from ⁇ 5% by volume to ⁇ 70% by volume, preferably in the range from ⁇ 15% by volume to ⁇ 50% by volume, based on the total volume of the mixture.
  • the nucleic acids are isolated by contacting the sample with a matrix based on one or more silicon oxide compounds such as silicon dioxide (silica), silicate, glass and/or silica gel and incubating it long enough for binding.
  • the matrix may be in the usual designs known from the prior art, such as, for example, in the form of particles, as membrane or filter. To facilitate their removal, preference is given to the particles having magnetic properties. Incubation times between 10 seconds and 30 minutes may be convenient for nucleic acids. Incubation times in a range from 1 minute to 20 minutes, in particular of approx. 10 minutes, have turned out to be advantageous.
  • Nucleic acids are isolated preferably by using magnetic particles having a gelatinous silica coating. Nucleic acids are isolated preferably by using magnetic particles having a gelatinous silica coating and having an average particle size in the range from ⁇ 1 ⁇ m to ⁇ 25 ⁇ m, preferentially in the range from ⁇ 5 ⁇ m to ⁇ 15 ⁇ m and particularly preferably in the range from ⁇ 6 ⁇ m to ⁇ 10 ⁇ m, preferentially with a narrow size distribution. Further preferably, nucleic acids are isolated by using magnetic particles having a gelatinous silica coating and having an average particle size in the range from ⁇ 1 ⁇ m to ⁇ 5 ⁇ m, preferably with a narrow size distribution.
  • the magnetic or magnetically attractable particles are particles having a magnetic core based on iron oxide, preferably selected from the group comprising magnetite (Fe 3 O 4 ), maghemite ( ⁇ -Fe 2 —O 3 ) and/or ferrites.
  • Magnetic silica particles which may be used in an advantageous manner are described, for example, in the international application WO 01/71732, the entire contents of which are hereby incorporated by reference.
  • a matrix based on one or more silicon oxide compound in the form of magnetic or magnetically attractable particles having a silica surface may be used.
  • the nucleic acids bound to the matrix based on one or more silicon oxide compound are removed from the lysis and/or binding composition.
  • magnetic silica particles this may be achieved with the aid of a magnetic field.
  • the magnetic particles may be drawn to the wall of the vessel in which incubation had been carried out, collected in suitable pipette tips by applying a magnetic field, or immobilized on magnetic rods protected by plastic coatings.
  • suitable method steps for removing the lysis and/or binding composition are removing by pipetting or aspirating the liquid or lifting the magnetic particles out in pipette tips or on magnetic rods, or lowering the lysis and/or binding mix, with the separated magnetic particles remaining at the same level.
  • the nucleic acid(s) immobilized on the matrix may be washed prior to removal.
  • the wash step is preferably carried out by incubating a wash solution with the loaded particles, involving preferably resuspension of said particles, for example by shaking or applying a magnetic field.
  • the contaminated wash solution is preferably removed, as is the lysis and/or binding composition remaining after binding, in particular a mixture of lysis composition and/or binding composition.
  • the wash reagent used may be a conventional wash buffer or any other suitable medium. Generally, preference is given to wash reagents having a low to moderate ionic strength, for example a solution of 10 mM tris(hydroxymethyl)aminomethane (TRIS). Furthermore usable are wash buffers having relatively high salt concentrations, for example a solution of 4-6 M guanidinium hydrochloride.
  • the wash reagents of the invention, described above, are likewise suitable wash reagents.
  • alcohol-containing wash reagents may also be used, for example aqueous solutions of alcohols having from one to five carbon atoms, preferably aqueous solutions of ethanol, in particular aqueous solutions of 50-100 percent strength ethanol.
  • washing is carried out first with wash reagents having a low to moderate ionic strength and then with an aqueous, 70-100 percent strength solution of ethanol.
  • using magnetic particles enables separation and/or wash steps to be carried out easily due to magnetic aggregation of the particles.
  • the last wash step or a water rinse may be followed by a step of drying the preferably magnetic particles, for example in vacuo or by evaporating the liquid or by letting the liquid evaporate.
  • the bound nucleic acids may be removed from the matrix. Removing the nucleic acids is also referred to us eluting.
  • the bound nucleic acid may be removed from the particles by means of an elution reagent having a low salt content.
  • reagents having a salt content of less than 0.1 mol/l may be used as the elution reagent having a low salt content.
  • Tris(hydroxymethyl)aminomethane (TRIS) particularly preferred for elution is furthermore demineralized water, optionally containing one or more additives, for example complexing agents such as ethylenediamine tetraacetate (EDTA), azide and/or buffer compounds, for example tris(hydroxymethyl)aminomethane (TRIS).
  • Using the lysis and/or binding composition results in a particularly advantageous method for isolating nucleic acids from biological samples, in particular for isolating viral DNA.
  • the present invention furthermore relates to a kit for isolating and/or purifying nucleic acids from a nucleic acid-containing biological sample, comprising a lysis, binding and/or wash reagent of the invention.
  • the kit may furthermore contain a matrix based on one or more silicon oxide compound, in particular a matrix based on one or more silicon oxide compound in the form of magnetic or magnetically attractable particles having a silica surface.
  • a matrix based on one or more silicon oxide compound in the form of magnetic or magnetically attractable particles having a silica surface. Examples of magnetic silica particles preferably contained in said kit are described in the international application WO 01/71732, the entire contents of which are hereby incorporated by reference.
  • the kit may furthermore contain suitable wash and/or elution reagents, in particular the wash reagent according to the invention.
  • the kit may contain silanized support materials other than magnetic silica particles, preferably centrifugal columns with silica membranes.
  • the present invention furthermore relates to the use of polyoxyethylene-based non-ionic surfactants selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers, in particular polyoxyethylene fatty alcohol ethers selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and/or polyoxyethylene oleyl ether, for solubilizing lipids of a biological sample.
  • lipid means for the purposes of the present invention water-insoluble or at least mostly water-insoluble natural substances.
  • lipid comprises for the purposes of the present invention fatty acids, the group of triglycerides comprising fats and oils, waxes, phospholipids, sphingolipids, liposaccharides, and the group of isoprenoids comprising steroids and carotenoids. More specifically, the term “lipid” means lipid components or structural components of the cell membranes of organisms, such as phospholipids and sphingolipids.
  • polyoxyethylene-based non-ionic surfactants selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers for solubilizing lipids of a biological sample in methods for isolating and/or purifying nucleic acids from a nucleic acid-containing biological sample.
  • polyoxyethylene fatty alcohol ethers selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and/or polyoxyethylene oleyl ether for solubilizing lipids of a biological sample in methods for isolating and/or purifying nucleic acids from a nucleic acid-containing biological sample.
  • polyoxyethylene-based non-ionic surfactants in particular polyoxyethylene fatty alcohol ethers, for solubilizing lipids of a biological sample in methods for isolating and/or purifying nucleic acids using a matrix based on one or more silicon oxide compound, preferably in the form of magnetic or magnetically attractable particles having a silica surface.
  • the eluate was found to contain no or at least markedly fewer contaminations when using the polyoxyethylene-based non-ionic surfactants, in particular polyoxyethylene fatty alcohol ethers, in methods for isolating and/or purifying nucleic acids using a matrix based on one or more silicon oxide compound, preferably in the form of magnetic or magnetically attractable particles having a silica surface.
  • polyoxyethylene-based non-ionic surfactants in particular polyoxyethylene fatty alcohol ethers
  • the invention concerns the use of polyoxyethylene-based non-ionic surfactants selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers, preferably polyoxyethylene fatty alcohol ether selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and/or polyoxyethylene oleyl ether, for preparing storage-stable binding, lysis and/or wash reagents.
  • polyoxyethylene-based non-ionic surfactants selected from the group comprising polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenyl ether, and/or polyoxyethylene-polyoxypropylene block copolymers, preferably polyoxyethylene fatty alcohol ether selected from the group comprising polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and/or polyoxyethylene oleyl
  • “Storage stable” here preferably means for the purposes of the invention that the properties of the lysis, binding or wash reagent that are relevant for the particular use, do not change during storage over a period of three months, preferably of 6 months, further preferably of at least 8 months, in such a way that this substantially impairs said use.
  • storage stability is displayed at room temperature, further preferably also at an elevated storage temperature of 50° C., for example.
  • the pH has turned out to be one of the properties of the reagents that are relevant to the use. Preferentially, therefore, the former does not substantially change during storage of the reagents, with the pH preferably decreasing by less than 1 during storage of the reagents.
  • FIG. 1 a , 1 b depict the change in pH of the lysis reagent B of the invention, depicted as empty bars, and of the Tween 20®-containing lysis reagent A, depicted as filled bars, during 33 weeks of storage at 25° C. ( FIG. 1 a ) and 50° C. ( FIG. 1 b ).
  • FIG. 2 depicts the averages of CT values after HBV-specific real time PCR of HBV-DNA after carrying out preparations of viral DNA with the lysis reagent B of the invention and the Tween® 20-containing lysis reagent A.
  • FIG. 3 depicts the averages of CT values after HBV-specific real time PCR of HBV-DNA after carrying out preparations of viral DNA with the lysis reagent B of the invention and the Tween® 20-containing lysis reagent A after 10 weeks of storage at 50° C.
  • a lysis reagent A which was stored at room temperature for about 4 weeks was used as reference.
  • 6 ⁇ l and 24 ⁇ l of the eluate were used for the real time PCR, with the results for 6 ⁇ l of the eluate being depicted as empty bars and the results for 24 ⁇ l of eluate being depicted as filled bars.
  • a lysis reagent A containing 20% (w/v) Tween® 20 (Fluka), guanidinium isothiocyanate and tris(hydroxymethyl)aminomethane and lysis reagent B in which Tween® 20 had been replaced with 20% (w/v) Brij® 58 (Sigma) were freshly prepared in double-distilled water and each stored in sealed vessels at 25° C. and 50° C. for 33 weeks.
  • the pH of each solution was determined here with the aid of a pH meter (Metrohm) at temperatures in the range from 20° C. to 28° C. at the start of storage and at weekly intervals.
  • a pH meter Mohm
  • the bar chart depicted in FIG. 1 a indicates that the pH of lysis reagent A decreased slightly from approx. pH 7.8 to pH 7.2 during 33 weeks of storage at 25° C., while the pH decreased from approx. pH 7.8 to approx. pH 5.9 during 33 weeks of storage at 50° C., as depicted in FIG. 1 b .
  • the pH of lysis reagent B remained stable at approx. pH 8 during 33 weeks of storage at 25° C. and 50° C.
  • Negative, i.e. HBV virus-free, human plasma was admixed with 10 4 sgU/ml hepatitis B virus (HBV).
  • the viral DNA was extracted from in each case 1000 ⁇ l of the plasma sample, using the commercially available automation platform QIAsymphony® (Qiagen), by means of the automated protocol for purifying viral nucleic acids from plasma samples.
  • the sample was exposed to the protocol-defined volumes—of lysis reagent B containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 20% (w/v) Brij® 58 (Sigma) and proteinase K, and solution AVE containing carrier RNA. This was followed by incubation at 65° C. to lyse the sample. To the sample mix was then added the protocol-defined volume of binding reagent C containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 9% (w/v) Brij® 58 (Sigma), and isopropanol.
  • MagAttract suspension which contained the magnetic silicon dioxide particles was added and mixed, as specified in the protocol. During this time, the nucleic acids bind to the silicon oxide particles. The magnetic silicon oxide particles were then separated and the liquid phase was removed. To the silicon oxide particles was then added the protocol-defined volume of wash solution containing guanidinium thiocyanate and ethanol, and the particles were suspended in the wash solution. The supernatant was again removed and a protocol-defined volume of wash solution containing Tris, NaCl and ethanol was added and a second wash step was carried out. After separating and removing the liquid phase, the particles were washed with protocol-defined volumes of aqueous 80% strength ethanol.
  • viral DNA was extracted from a further 1000 ⁇ l of plasma sample, with the modification of using lysis reagent A containing 20% (w/v) Tween® 20 (Fluka) and binding reagent D containing 9% (w/v) Tween® 20 (Fluka).
  • the eluates obtained were subjected in each case to an HBV-specific real time (RT-) PCR, with 24 ⁇ l of eluate being used in each case.
  • RT- real time
  • the averages, depicted in FIG. 2 of the CT values (threshold cycle) which describe the cycle at which the fluorescence begins to increase logarithmically revealed that the extraction using the lysis reagent B and binding reagent C of the invention achieved a higher yield.
  • the lysis reagents B containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 20% (w/v) Brij® 58 (Sigma), and A in which Brij® 58 had been replaced with 20% (w/v) Tween® 20 (Fluka) were each stored in sealed vessels at 50° C. for 10 weeks.
  • the viral nucleic acid was then extracted, using the commercially available automation platform QIAsymphony® (Qiagen), by means of the automated protocol for purifying viral nucleic acid from plasma samples.
  • the viral DNA was extracted from in each case 1000 ⁇ l of the plasma sample using the commercially available automation platform QIAsymphony® (Qiagen), with, for different mixtures, lysis reagent B containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 20% (w/v) Brij® 58 (Sigma), and lysis reagent A in which Brij® 58 had been replaced with 20% (w/v) Tween® 20 (Fluka) being stored in each case at 50° C. for 10 weeks.
  • QIAsymphony® Qiagen
  • the reference used was a lysis reagent A which was stored at room temperature for about 4 weeks.

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US20220002708A1 (en) 2022-01-06
JP2017029160A (ja) 2017-02-09
CA2725586A1 (en) 2009-12-03
JP6499015B2 (ja) 2019-04-10
JP7069059B2 (ja) 2022-05-17
KR102207033B1 (ko) 2021-01-22
MX2010012849A (es) 2011-03-29
CA2725586C (en) 2019-05-07
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